Method for producing an organic acid

ABSTRACT

An organic acid is produced by allowing a bacterium which has an ability to produce an organic acid and has been modified so that expression of the sucE1 and mdh genes are enhanced, or a product obtained by processing the bacterium, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and collecting the organic acid.

This application is a continuation under 35 U.S.C. §120 of PCT PatentApplication No. PCT/JP2008/057086, filed Apr. 10, 2008, which claimspriority under 35 U.S.C. §119 to Japanese Patent Application No.2007-102668, filed on Apr. 10, 2007, which are incorporated in theirentireties by reference. The Sequence Listing in electronic format filedherewith is also hereby incorporated by reference in its entirety (FileName: US-410_Seq_List; File Size: 91 KB; Date Created: Oct. 9, 2009).

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for producing an organic acidusing a bacterium such as a coryneform bacterium.

2. Background Art

For the production of non-amino organic acids, including succinic acid,by fermentation, anaerobic bacteria are typically used, includinganaerobic bacteria belonging to the genus Anaerobiospirillum orActinobacillus (U.S. Pat. No. 5,142,834, International Journal ofSystematic Bacteriology, 49, 207-216 (1999)). Although such anaerobicbacteria provide high yields of products, many nutrients are requiredfor their proliferation, and therefore it is necessary to add largeamounts of organic nitrogen sources such as corn steep liquor (CSL) intothe culture medium. The addition of large amounts of sources of organicnitrogen results in not only an increase in cost for the culture medium,but also an increase in the purification costs for isolating theproduct, and therefore it is not economical.

In addition, methods are known in which aerobic bacteria such ascoryneform bacteria are cultured once under aerobic conditions toproliferate the bacterial cells, then the cells are harvested, washed,and allowed to rest so that a non-amino organic acid is produced withouthaving to supply oxygen (Japanese Patent Laid-open (KOKAI) Nos.11-113588 and 11-196888). When aerobic bacteria which were grown underaerobic conditions are used for the production of non-amino organicacids as described above, a culture under microaerobic conditions (alsocalled “microaerobic induction”) is generally necessary to producesuccinic acid under anaerobic conditions. These methods are economical,since organic nitrogen can be added in a smaller amount forproliferation of the bacterial cells, and the bacteria can sufficientlygrow in a simple culture medium. However, there is still a room forimprovement in terms of production amounts, concentration, andproduction rate per cell of the target organic acids, as well assimplification of the production process, and the like. Furthermore,techniques of increasing a non-amino organic acid-producing ability byDNA recombination have also been disclosed. For example, production of anon-amino organic acid by fermentation using a bacterium in whichphosphoenolpyruvate carboxylase activity is enhanced (for example,Japanese Patent Laid-open No. 11-196887), and the like, have also beenreported.

The entire genome sequence of a coryneform bacterium has been reported,and the functions of putative protein-coding sequences have beenpredicted (Appl. Microbiol. Biotechnol., 62 (2-3), 99-109 (2003)). ThesucE1 gene is one of these putative protein-coding sequences, andalthough the gene is thought to code for a permease, the actual functionhas not been clarified. Finally, participation of the sucE1 gene in thesuccinic acid synthetic pathway was also not known. As for maleatedehydrogenase, although a method for producing succinic acid using abacterium in which activity of this enzyme is enhanced has been reported(Japanese Patent Laid-open No. 2006-320208), the effect of enhancing themdh gene along with the sucE1 gene has not been reported

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a method for producingan organic acid using a bacterium using a simplified production process.

It has been found that a bacterium which has been modified to enhanceexpression of the sucE1 and mdh genes can produce an organic acid froman organic raw material without the need for microaerobic induction.

It is an aspect of the present invention to provide a method forproducing an organic acid comprising: A) allowing a substance to act onan organic raw material in a reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas, wherein the substance isselected from the group consisting of: i) a bacterium which has anability to produce an organic acid and has been modified to haveenhanced expression of sucE1 and mdh genes, ii) a product obtained byprocessing the bacterium of i), and iii) combinations thereof, and B)collecting the organic acid.

It is a further aspect of the present invention to provide theaforementioned method, wherein expression of the sucE1 and mdh genesunder aerobic conditions is increased by 1.5 times or more as comparedto an unmodified strain.

It is a further aspect of the present invention to provide theaforementioned method, wherein the bacterium is selected from the groupconsisting of coryneform bacteria, Bacillus bacteria, and Rhizobiumbacteria.

It is a further aspect of the present invention to provide theaforementioned method, wherein enhanced expression is obtained by amethod selected from the group consisting of i) increasing the copynumber of, ii) modifying an expression control sequence of the sucE1gene and/or the mdh gene, iii) replacing a promoter of the sucE1 geneand/or the mdh gene with a stronger promoter, and iv) combinationsthereof.

It is a further aspect of the present invention to provide theaforementioned method, wherein the stronger promoter is a constitutiveexpression promoter.

It is a further aspect of the present invention to provide theaforementioned method, wherein the stronger promoter is a promoter of agene that encodes a protein selected from the group consisting ofelongation factor Tu, cochaperonin GroES-chaperonin GroEL, thioredoxinreductase, phosphoglycerate mutase, peroxiredoxin, glycerol-3-phosphatedehydrogenase, 2,3-butanediol dehydrogenase, fructose bisphosphatealdolase, and superoxide dismutase.

It is a further aspect of the present invention to provide theaforementioned method, wherein the sucE1 gene is selected from the groupconsisting of: (a) a DNA comprising the nucleotide sequence of thenumbers 571 to 2187 of SEQ ID NO: 3, and (b) a DNA which hybridizes witha nucleotide sequence complementary to the nucleotide sequence of thenumbers 571 to 2187 of SEQ ID NO: 3 under stringent conditions, and theDNA improves the ability of the bacterium to produce succinic acid whenexpression of the DNA is enhanced in the bacterium.

It is a further aspect of the present invention to provide theaforementioned method, wherein the mdh gene is selected from the groupconsisting of: (a) a DNA comprising the nucleotide sequence of thenumbers 301 to 1287 of SEQ ID NO: 13, and (b) a DNA which hybridizeswith a nucleotide sequence complementary to the nucleotide sequence ofthe numbers 301 to 1287 of SEQ ID NO: 13 under stringent conditions, andcodes for a protein having malate dehydrogenase activity.

It is a further aspect of the present invention to provide theaforementioned method, wherein the bacterium has been further modifiedso that lactate dehydrogenase activity is decreased to 10% or less ofthe activity as compared to lactate dehydrogenase activity in anunmodified strain.

It is a further aspect of the present invention to provide theaforementioned method, wherein the bacterium has been further modifiedso that pyruvate carboxylase activity is enhanced.

It is a further aspect of the present invention to provide theaforementioned method, wherein the organic acid is succinic acid.

It is a further aspect of the present invention to provide a method forproducing a succinic acid-containing polymercomprising: A) producingsuccinic acid by the aforementioned method, and B) polymerizing thesuccinic acid.

It is a further aspect of the present invention to provide theaforementioned method, wherein the bacterium has been further modifiedso that succinate dehydrogenase activity is enhanced.

It is a further aspect of the present invention to provide theaforementioned method, wherein the organic acid is malic acid or fumaricacid.

It is a further aspect of the present invention to provide theaforementioned method, wherein the bacterium has been further modifiedso that succinate dehydrogenase activity is decreased to 10% or less ascompared to succinate dehydrogenase activity in an unmodified strain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction of plasmid pBS4SΔldh56, which is used for ldhgene disruption.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, embodiments of the present invention will be explained indetail.

Bacteria

The bacterium used in the method of the presently disclosed subjectmatter (also referred to as “the bacterium of the presently disclosedsubject matter”) can be a bacterium which has an ability to produce anorganic acid and has been modified so that expression of both the sucE1gene and the mdh gene are enhanced. This bacterium can be obtained bymodifying a bacterium having an organic acid-producing ability as aparent strain so that expression of both the sucE1 gene and the mdh genein the bacterium are simultaneously enhanced. When the parent strain isnot able to produce an organic acid, the bacterium can be obtained byimparting this ability to the parent strain, and then modifying thestrain so that expression of both the sucE1 gene and the mdh gene areenhanced. Furthermore, the bacterium can also be obtained by impartingan organic acid-producing ability to a strain which has previously beenmodified so that expression of both the sucE1 gene and the mdh gene areenhanced.

The organic acid can be a metabolic intermediate of the TCA cycle, andexamples include succinic acid, malic acid, fumaric acid, citric acid,isocitric acid, cis-aconitic acid, and the like.

Parent strains that can be used to derive the bacteria of the presentlydisclosed subject matter are not particularly limited. However, aerobicbacteria and facultative anaerobic bacteria are examples, specifically,coryneform bacteria, Bacillus bacteria, Rhizobium bacteria, andEscherichia bacteria are also examples. Examples of coryneform bacteriainclude microorganisms belonging to the genus Corynebacterium,microorganisms belonging to the genus Brevibacterium, and microorganismsbelonging to the genus Arthrobacter. Among these, those belonging to thegenus Corynebacterium or Brevibacterium are examples. Microorganismsbelonging to Corynebacterium glutamicum, Brevibacterium flavum,Brevibacterium ammoniagenes, or Brevibacterium lactofermentum are otherexamples.

Specific examples of the aforementioned parent strains of bacteriainclude Brevibacterium flavum MJ-233 (Agric. Biol. Chem., 54 (2),443-447, 1990), MJ-233 AB-41, which is a mutant strain of MJ-233(Japanese Patent Laid-open No. 2003-235592), Corynebacterium glutamicum(Corynebacterium sp., Brevibacterium flavum) AJ110655 (PERM ABP-10951),Brevibacterium ammoniagenes ATCC 6872, Corynebacterium glutamicum ATCC31831, ATCC 13032, Brevibacterium lactofermentum ATCC 13869, and thelike. Since Brevibacterium flavum can be currently classified intoCorynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. andSchleifer, K. H., International Journal of Systematic Bacteriology,1991, vol. 41, pp. 255-260), the Brevibacterium flavum MJ-233 strain,MJ-233 AB-41 strain, and AJ110655 strain are considered to be identicalto the Corynebacterium glutamicum MJ-233 strain, MJ-233 AB-41 strain,and AJ110655 strain, respectively.

The Corynebacterium glutamicum AJ110655 strain was deposited at theInternational Patent Organism Depository, Agency of Industrial Scienceand Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, 305-8566) on Feb. 15, 2008 and assigned a receiptnumber of FERM ABP-10951. The Corynebacterium glutamicum AJ110655 is anLDH gene-deficient strain constructed from the Brevibacterium flavumMJ-233 strain, and was deposited as the Corynebacterium glutamicumAJ110655 strain. Then, it was suggested that the species thereof wasdifferent from Corynebacterium glutamicum on the basis of nucleotidesequence analysis of 16S rRNA etc.

The parent strain which can be used to obtain the bacterium inaccordance with the presently disclosed subject matter can be, besideswild-type strains, any strain, including mutant strains obtained bytypical mutation treatments such as UV irradiation and NTG treatment,and recombinant strains induced by genetic procedures such as cellfusion and gene recombination techniques.

When the parent strain is not able to produce organic acids, thisability can be imparted by mutation or gene recombination. However, whenthe organic acid-producing ability is imparted by enhancing expressionof the sucE1 gene and the mdh gene, it is not always necessary to impartthe organic acid-producing ability by other means.

The “organic acid-producing ability” refers to an ability of thebacterium to cause accumulation of an organic acid in a reaction mixturecontaining carbonate ions, bicarbonate ions, or carbon dioxide to suchan extent that the organic acid can be collected from the reactionmixture when the bacterium, or a product obtained by processing thebacterium, is allowed to act on a organic raw material in the reactionmixture.

The methods of imparting an organic acid-producing ability to amicroorganism will be specifically explained below.

Succinic Acid-Producing Bacteria

Examples of the method for imparting or enhancing succinicacid-producing ability by breeding include, for example, modifying abacterium so that expression of a gene coding for an enzyme involved insuccinic acid biosynthesis is enhanced. Examples of enzymes involved insuccinic acid biosynthesis include, for example, pyruvate carboxylase,fumarate reductase (Japanese Patent Laid-open No. 2005-095169), and thelike. Bacteria in which the pyruvate carboxylase and fumarate reductasegenes are amplified are described in Japanese Patent Laid-open based onInternational Patent Application (Kohyo) No. 2002-511250, JapanesePatent Laid-open Nos. 11-196888, 2005-95169, and the like.

Succinic acid-producing ability can also be imparted or enhanced bydisrupting a gene coding for lactate dehydrogenase, which is an enzymethat is expressed under anaerobic conditions as described herein.Succinic acid-producing ability can also be imparted by treating abacterium with ultraviolet rays or with a mutagen typically employed inmutation treatments such as N-methyl-N-nitro-N-nitrosoguanidine (NTG) orethyl methanesulfonate (EMS), and selecting a strain which producessuccinic acid. Examples of mutants having succinic acid-producingability include the glutamic acid-auxotrophic strain disclosed inJapanese Patent Laid-open No. 2005-065641, and the like.

The bacterium can be obtained by modifying such a bacterium havingsuccinic acid-producing ability as described above so that expression ofthe sucE1 gene and the mdh gene are enhanced. Either the modificationfor imparting succinic acid-producing ability or the modification forenhancing expression of the sucE1 gene and the mdh gene can be performedfirst.

sucE1 Gene

The protein encoded by the sucE1 gene (SucE, succinate exporter) ispresumed to be a kind of permease, and is a protein which improvessuccinic acid-producing ability of a bacterium when expression of thegene is enhanced in the bacterium.

Examples of the sucE1 gene include the sucE1 gene derived from or nativeto Brevibacterium flavum MJ-233 (nucleotide numbers 571 to 2187 of SEQID NO: 3), the sucE1 gene derived from or native to Corynebacteriumglutamicum ATCC 13032 (SEQ ID NO: 5), the sucE1 gene derived from ornative to Corynebacterium efficiens YS314 (SEQ ID NO: 7), the sucE1 genederived from or native to Corynebacterium diphtheriae gravis NCTC 13129(SEQ ID NO: 11), and the like. The sucE1 gene derived from C. glutamicumATCC 13032 is registered as NCg12130 in the genome sequence registeredas GenBank Accession No. NC_(—)003450 (amino acid sequence is registeredas GenBank Accession No. NP_(—)601414). The sucE1 gene derived from ornative to C. efficiens YS314 is registered as CE2102 in the genomesequence registered as GenBank Accession No. NC_(—)004369. The sucE1gene derived from or native to C. diphtheriae gravis NCTC 13129 isregistered as D1P0830 of GenBank Accession No. NC_(—)002935.

A homologue gene of sucE1 derived from another microorganism can be usedas the sucE1 gene so long as the homologous gene can improve succinicacid-producing ability of a bacterium when its expression is enhanced inthe bacterium.

Homologues of the sucE1 gene can be searched for by using BLAST(//blast.genome.jp/), or the like, by referring to the sequence of thenucleotide numbers 571 to 2187 of SEQ ID NO: 3, and the like.

Since several sequences of the sucE1 gene have already been determined,the gene can be obtained by PCR using primers prepared on the basis ofthe nucleotide sequences. For example, a region including the structuralsucE1 gene of C. glutamicum and a flanking region thereof including acontrol region of the gene can be obtained by PCR (polymerase chainreaction, see White, T. J. et al., Trends Genet., 5, 185 (1989)) usingthe primers shown in SEQ ID NOS: 1 and 2 and chromosomal DNA of acoryneform bacterium as the template. Homologues of sucE1 of othermicroorganisms can also be obtained in a similar manner.

Since the nucleotide sequence of the sucE1 gene is different dependingon the species or strains of coryneform bacteria, the sucE1 gene is notlimited to a gene having the sequence of the nucleotide numbers 571 to2187 of SEQ ID NO: 3 or the sequence of SEQ ID NO: 5, 7 or 11, but itcan also be a mutant or artificially modified gene that codes for aprotein having a sequence of SEQ ID NO: 4, 6, 8 or 12 but which includessubstitutions, deletions, insertions, additions, etc. of one or severalamino acid residues at one or more positions. However, this mutated orartificially modified gene can still improve succinic acid-producingability of the bacterium when expression of the gene is enhanced in thebacterium. Although the number meant by the term “several” in relationto the number of amino acid residues can differ depending on theposition in the three-dimensional structure of the protein or the typeof amino acid residue, it can be specifically 1 to 20, 1 to 10 inanother example, or 1 to 5 in another example. The substitutions,deletions, insertions, additions, inversions or the like of amino acidresidues described above can also include those caused by a naturallyoccurring mutation based on individual difference, difference in speciesof microorganisms from which the sucE1 gene is derived (mutant orvariant), or the like.

The aforementioned substitution can be a conservative substitution thatis a neutral substitution, that is, not resulting in a functionalchange. The conservative mutation is a mutation wherein substitutiontakes place mutually among Phe, Trp and Tyr, if the substitution site isan aromatic amino acid; among Leu, Ile and Val, if the substitution siteis a hydrophobic amino acid; between Gln and Asn, if it is a polar aminoacid; among Lys, Arg and His, if it is a basic amino acid; between Aspand Glu, if it is an acidic amino acid; and between Ser and Thr, if itis an amino acid having hydroxyl group. Specific examples ofsubstitutions considered to be conservative substitutions can include:substitution of Ser or Thr for Ala; substitution of Gln, His or Lys forArg; substitution of Glu, Gln, Lys, His or Asp for Asn; substitution ofAsn, Glu or Gln for Asp; substitution of Ser or Ala for Cys;substitution of Asn, Glu, Lys, His, Asp or Arg for Gln; substitution ofGly, Asn, Gln, Lys or Asp for Glu; substitution of Pro for Gly;substitution of Asn, Lys, Gln, Arg or Tyr for His; substitution of Leu,Met, Val or Phe for Ile; substitution of Ile, Met, Val or Phe for Leu;substitution of Asn, Glu, Gln, His or Arg for Lys; substitution of Ile,Leu, Val or Phe for Met; substitution of Trp, Tyr, Met, Ile or Leu forPhe; substitution of Thr or Ala for Ser; substitution of Ser or Ala forThr; substitution of Phe or Tyr for Trp; substitution of His, Phe or Trpfor Tyr; and substitution of Met, Ile or Leu for Val.

Furthermore, as the sucE1 gene, a sequence encoding a protein having ahomology not less than 80% in one example, not less than 90% in anotherexample, not less than 95% in another example, or not less than 97% inanother example, to the entire amino acid sequence of SEQ ID NOS: 4, 6,8 or 12 and coding for a protein which improves succinic acid-producingability of a bacterium when expression is enhanced in the bacterium canbe used. Furthermore, the degree of degeneracy of a gene variesdepending on the host into which the gene is introduced, and thereforecodons can be replaced with those which are favorable for the chosenhost of the sucE1. Moreover, the sucE1 gene can encode for a proteinwith an elongated or deleted N- or C-terminal sequence, so long as thegene improves the succinic acid-producing ability of a bacterium whenexpression is enhanced in the bacterium. The length of amino acidsequence to be elongated or deleted can be 50 or less, 20 or less inanother example, 10 or less in another example, 5 or less in anotherexample, in terms of number of amino acid residues. More specifically,the sucE1 gene can encode for a protein having the amino acid sequenceof SEQ ID NO: 4, 6, 8 or 12 with elongation or deletion of 5 to 50 aminoacid residues on the N-terminal or C-terminal side.

Genes which are homologous to the sucE1 gene as described above can beobtained by modifying the nucleotide sequence of nucleotide numbers 571to 2187 of SEQ ID NO: 3, or the nucleotide sequence of SEQ ID NO: 5, 7or 11 so that the protein encoded by the gene includes substitutions,deletions, insertions, or additions of amino acid residues at a specificsite(s) by, for example, site-specific mutagenesis. Furthermore,homologous genes can also be obtained by conventionally known mutationtreatments, such as those described below. Examples of mutationtreatments include treating the nucleotide sequence of nucleotides 571to 2187 of SEQ ID NO: 3, or the nucleotide sequence of SEQ ID NO: 5, 7or 11, with hydroxylamine, or the like, in vitro, and treating amicroorganism, for example, a coryneform bacterium, containing the genewith ultraviolet ray irradiation or a mutagen typically used formutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS). Furthermore, a mutation can be artificiallyintroduced into the sucE1 gene by gene recombination using error-pronePCR, DNA shuffling, or StEP-PCR to obtain a highly active sucE1 gene(Firth A. E., Patrick W. M., Bioinformatics., 21, 3314-3315, 2005 Jun.2; Statistics of protein library construction).

Whether such homologous sucE1 genes code for a protein which improvessuccinic acid-producing ability when expression is enhanced can beconfirmed by, for example, introducing these genes into a wild-typestrain of a bacterium and determining whether the succinicacid-producing ability of the bacterium is improved or not.

Examples of the sucE1 gene also include a DNA that hybridizes with asequence complementary to the sequence of nucleotides 571 to 2187 of SEQID NO: 3, the nucleotide sequence of SEQ ID NO: 5, 7 or 11, or a probethat can be prepared from the sequences under stringent conditions andcodes for a protein which improves succinic acid-producing ability of abacterium when expression is enhanced in the bacterium. The “stringentconditions” referred to herein are conditions under which a so-calledspecific hybrid is formed, and non-specific hybrid is not formed.Examples include, for example, conditions where DNAs showing highhomology to each other, for example, DNAs showing a homology of, forexample, not less than 80%, not less than 90% in another example, notless than 95% in another example, or not less than 97% in anotherexample, hybridize with each other, and DNAs having homology lower thanthe above level do not hybridize with each other. Other examples includetypical washing conditions in Southern hybridization, i.e., washingonce, twice or three times, at salt concentrations and temperature of1×SSC, 0.1% SDS at 60° C., or 0.1×SSC, 0.1% SDS at 60° C., or 0.1×SSC,0.1% SDS at 68° C. in another example.

A partial sequence of the nucleotides 571 to 2187 of SEQ ID NO: 3 or apartial sequence of SEQ ID NO: 5, 7 or 11 can also be used as the probe.Such a probe can be prepared by PCR using oligonucleotides prepared onthe basis of these nucleotide sequences as primers and a DNA fragmentcontaining the sequence as the template. When a DNA fragment having alength of about 300 by is used as the probe, the washing conditionsafter hybridization under the aforementioned conditions can beexemplified by 2×SSC, 0.1% SDS at 50° C.

The expression “modified so that expression of sucE1 gene is enhanced”means that the number of SucE1 protein molecules per cell is increased,or that the activity per SucE1 protein molecule is increased, etc., ascompared to an unmodified strain such as a parent strain or a wild-typestrain. Examples of the wild-type coryneform bacterium which can be usedfor comparison include Corynebacterium glutamicum (Brevibacteriumlactofermentum) ATCC 13869, ATCC 13032, and the like (the same shallapply to the other genes mentioned below).

Increase of expression level of the sucE1 gene can be confirmed bycomparing the level of mRNA of sucE1 with that of an unmodified strainsuch as a parent strain or a wild-type strain. Examples of the methodfor confirming the expression level include Northern hybridization andRT-PCR (Molecular Cloning (Cold Spring Harbor Laboratory Press, ColdSpring Harbor (USA), 2001). The expression level of sucE1 can be anylevel so long as the level is increased as compared to that of anunmodified strain, and for example, it can be increased not less than1.5 times, not less than 2 times in another example, or not less than 3times in another example, as compared to that of an unmodified strain.The term “aerobic conditions” can mean that the dissolved oxygenconcentration is not lower than 0.33 ppm and not higher than 7 ppm, ornot lower than 1.5 ppm and not higher than 7 ppm in another example(Amino Acid Fermentation, Kunihiko Akashi et al.).

mdh Gene

The mdh gene is not particularly limited so long as it encodes for aprotein having malate dehydrogenase activity, and examples include, forexample, the gene derived from the Brevibacterium flavum MJ-233 strainhaving the nucleotide sequence shown in SEQ ID NO: 13. The phrase“malate dehydrogenase activity” can mean the activity for catalyzing thereaction of reducing oxaloacetic acid to malic acid, and the expression“malate dehydrogenase activity is enhanced” means that the malatedehydrogenase activity is enhanced as compared to that of a strain inwhich malate dehydrogenase is unmodified.

A homologue gene of mdh derived from another microorganism can be usedas the mdh gene so long as the homologue gene codes for a protein havingmalate dehydrogenase activity. Homologues of the mdh gene can besearched for by using BLAST (http://blast.genome.jp/), or the like, byreferring to the sequence of nucleotides 301 to 1384 of SEQ ID NO: 13.

Since several sequences of the mdh gene have already been determined asdescribed above, homologous genes can be obtained by PCR using primersprepared on the basis of the known nucleotide sequences. For example, aregion including the structural mdh gene of C. glutamicum and flankingregions, which include a control region of the gene, can be obtained byPCR using the primers shown in SEQ ID NOS: 15 and 16 and chromosomal DNAof the coryneform bacterium as the template. Homologues of mdh fromother microorganisms can also be obtained in a similar manner.

The foregoing descriptions concerning mutants or artificially modifiedhomologues of the sucE1 gene and protein are also applicable to the mdhgene. These descriptions are also applicable to the LDH gene and PC genedescribed later.

The expression “modified so that expression of mdh gene is enhanced” canmean that the bacterium is modified so that the activity of malatedehydrogenase encoded by the mdh gene is enhanced as compared to an mdhunmodified strain, such as a parent strain or a wild-type strain. Thisexpression can also include when the number of malate dehydrogenasemolecules per cell is increased, or when the activity per maleatedehydrogenase molecule is increased, etc. Examples of the wild-typecoryneform bacterium which can be used for comparison includeCorynebacterium glutamicum (Brevibacterium lactofermentum) ATCC 13869,ATCC 13032, and the like.

An increase in the expression of the mdh gene can be confirmed bycomparing the level of the mdh mRNA with that of an unmodified strain asdescribed above. An increase in the expression level of the mdh gene canalso be confirmed by comparing the malate dehydrogenase activity withthat of an unmodified strain.

The expression level of the mdh gene can be increased to any level solong as it is increased as compared to that of an unmodified strainunder aerobic conditions, and for example, it can be increased not lessthan 1.5 times, and not less than 2 times in another example, ascompared to an unmodified strain. The malate dehydrogenase activity canbe measured by measuring the decrease of NADH as described later.

Enhancing Expression of the sucE1 and mdh Genes

Expression of the sucE1 gene and the mdh gene can be enhanced byincreasing the copy numbers of these genes. For example, the copynumbers of the genes can be increased by ligating a fragment containingthe genes to a vector which functions in the chosen bacterium, forexample, a multi copy vector, to prepare a recombinant DNA, andtransforming the bacterium as described above with the DNA.Alternatively, the copy numbers of the genes can be increased bytransferring one copy or multiple copies of the genes to the bacterialchromosome. Transfer of the genes to the chromosome can be confirmed bySouthern hybridization using a part of the genes as a probe.

When a plasmid vector is used, the sucE1 gene and the mdh gene can becarried on the same plasmid, or can be separately carried on differentplasmids. Moreover, the order in which the sucE1 gene and the mdh geneare introduced into the bacterium is not limited, and they can beintroduced simultaneously.

Expression of the sucE1 gene and the mdh gene can also be enhanced bymodifying an expression control sequence of these genes. For example, apromoter sequence of the genes can be replaced with a stronger promoter,or the promoter sequence can be brought closer to a consensus sequence(WO00/18935).

Methods for modifying a microorganism so that expression of the sucE1gene and the mdh gene are enhanced are specifically explained below.These methods can be performed as described in a manual such asMolecular Cloning (Cold Spring Harbor Laboratory Press, Cold SpringHarbor (USA), 2001).

First, a target gene can be cloned from the chromosome of a coryneformbacterium or the like. A chromosomal DNA can be prepared from abacterium by, for example, the method of Saito and Miura (see H. Saitoand K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Text forBioengineering Experiments, Edited by the Society for Bioscience andBioengineering, Japan, p 97-98, Baifukan Co., Ltd., 1992), or the like.Oligonucleotides for use in PCR can be synthesized on the basis of theaforementioned known information, for example, the syntheticoligonucleotides shown ins SEQ ID NOS: 1 and 2 can be used to amplifythe sucE1 gene, and the synthetic oligonucleotides shown in SEQ ID NOS:15 and 16 can be used to amplify the mdh gene.

A gene fragment which includes a gene amplified by PCR can itself beamplified by inserting the fragment into a vector having a replicationorigin that enables autonomous replication in the chosen bacterium, thentransform the bacterium with the vector. In particular, when using acoryneform bacterium as the host, if a recombinant DNA is prepared byligating the fragment to a vector DNA that is autonomously replicable incells of Escherichia coli and a coryneform bacterium, and introducedinto Escherichia coli, subsequent operations becomes easier. Examples ofvectors that are autonomously replicable in cells of Escherichia coliinclude pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322,pACYC184, pMW219, and the like.

Examples of plasmids that are autonomously replicable in coryneformbacteria include plasmids pCRY30 (described in Japanese Patent Laid-openNo. 3-210184); plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, andpCRY3KX (described in Japanese Patent Laid-open No. 2-72876 and U.S.Pat. No. 5,185,262); plasmids pCRY2 and pCRY3 (described in JapanesePatent Laid-open No. 1-191686); pAM330 (described in Japanese PatentLaid-open No. 58-67679); pHM1519 (described in Japanese Patent Laid-openNo. 58-77895); pAJ655, pAJ611, and pAJ1844 (described in Japanese PatentLaid-open No. 58-192900); pCG1 (described in Japanese Patent Laid-openNo. 57-134500); pCG2 (described in Japanese Patent Laid-open No.58-35197); pCG4, pCG11 etc. (described in Japanese Patent Laid-open No.57-183799); and pVK7 (described in Japanese Patent Laid-open No.10-215883).

Furthermore, a vector obtained by excising a DNA fragment that enables aplasmid to autonomously replicate in coryneform bacteria from any of theabove-listed vectors, and inserting the fragment into any of theaforementioned vectors for Escherichia coli can be used as a so-calledshuttle vector, which is autonomously replicable both in Escherichiacoli and coryneform bacteria.

To prepare a recombinant DNA by ligating the sucE1 gene and the mdh geneto a vector which functions in coryneform bacteria, the vector isdigested with a restriction enzyme suitable for the ends of the genes.Such a restriction enzyme site can be introduced in advance into thesynthetic oligonucleotide which is used to amplify the genes. Ligationis usually performed by using a ligase such as T4 DNA ligase.

In order to introduce a recombinant plasmid prepared as described aboveinto a bacterium, any known transformation method reported to date canbe employed. For example, recipient cells can be treated with calciumchloride so as to increase permeability for the DNA; this method hasbeen reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J.Mol. Biol., 53, 159 (1970)). Also, competent cells can be prepared fromgrowing cells and DNA can be introduced into these cells; this methodhas been reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A.and Young, F. E., Gene, 1, 153 (1977)). Another method is to make DNArecipient cells into protoplasts or spheroplasts which easily take up arecombinant DNA, and a recombinant DNA can be introduced into thesecells; this method is known for Bacillus subtilis, actinomycetes, andyeasts (Chang, S, and Choen, S. N., Mol. Gen. Genet., 168, 111 (1979);Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978);Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci. USA,75, 1929 (1978)). In addition, bacteria can also be transformed by theelectric pulse method (Japanese Patent Laid-open No. 2-207791) or by theconjugal transfer method (Biotechnology (NY). 1991 January; 9(1):84-7).

Copy numbers of the sucE1 and mdh genes can also be increased byintegrating multiple copies of the genes into the chromosomal DNA of abacterium, which can be accomplished by homologous recombination. Thistechnique is performed by targeting a sequence which is present inmultiple copies on the chromosomal DNA. Sequences present on thechromosomal DNA in multiple copies include repetitive DNA or invertedrepeats present at the end of a transposable element. Alternatively, asdisclosed in Japanese Patent Laid-open No. 2-109985, multiple copies ofthe genes can be introduced into a chromosomal DNA by incorporating theminto a transposon and transferring it (Japanese Patent Laid-open Nos.2-109985, 7-107976, Mol. Gen. Genet., 245, 397-405 (1994); Plasmid, 2000November; 44(3): 285-91).

Another possible method is to insert the sucE1 gene and/or the mdh geneinto a plasmid which has a replication origin that is not replicable orcannot replicate in the host, and which is able to cause conjugaltransfer to the host, and introducing this plasmid into the host toamplify the gene on the chromosome. Examples of such a plasmid includepSUP301 (Simo et al., Bio/Technology 1, 784-791 (1983)), pK18mob orpK19mob (Schaefer et al., Gene 145, 69-73 (1994)), pGEM-T (Promegacorporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994) Journal ofBiological Chemistry 269: 32678-84; U.S. Pat. No. 5,487,993), pCR^((R))Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991,Journal of Bacteriology 173: 4510-4516), pBGS8 (Spratt et al., 1986,Gene, 41:337-342), and the like. A plasmid vector which includes thesucE1 gene and/or the mdh gene is transferred into the bacterium byconjugation or transformation to transfer the genes onto the bacterialchromosome. The conjugation method is described by, for example,Schaefer et al. (Applied and Environmental Microbiology, 60, 756-759(1994)). The transformation method is described by, for example,Theirbach et al. (Applied Microbiology and Biotechnology 29, 356-362(1988)), Dunican and Shivinan (Bio/Technology 7, 1067-1070 (1989)), andTauch et al. (NEMS Microbiological Letters 123, 343-347 (1994)).Expression of sucE1 and mdh can also be enhanced by replacing the nativeexpression control sequences, such as promoters, of sucE1 and mdh on thechromosomal DNA or a plasmid with stronger promoters. For example, thelac promoter, trp promoter, trc promoter, PS2 promoter, and the like areknown as strong promoters. Methods for evaluating the strength ofpromoters and examples of strong promoters are described in the paper ofGoldstein et al. (Prokaryotic promoters in Biotechnology, Biotechnol.Annu. Rev., 1995, 1, 105-128), and the like. The strong promoter can bea constitutive expression promoter. The constitutive expression promotercan mean a promoter the expression of which is not changed bymicroaerobic induction. The term “microaerobic induction” can indicate aculture under microaerobic conditions, which imparts an activity ofproducing an organic acid under anaerobic conditions to cells whichtypically grow under aerobic conditions. The microaerobic conditionscorresponds to 2 ppm or lower, 1 ppm or lower in another example, or 0.5ppm or lower in another example, in terms of dissolved oxygenconcentration with or without aeration. Moreover, the anaerobiccondition is when the dissolved oxygen concentration is 0.5 ppm or lowerwhen bubbling a gas other than oxygen, such as nitrogen or carbondioxide.

The phrase “expression is not changed by microaerobic induction” meansthat the change in the ratio of the promoter activities observed beforeand after microaerobic induction is 3 times or smaller, 2 times orsmaller in another example, or 1.5 times or smaller in another example.Specific examples of the constitutive expression promoter includepromoters of genes coding for the elongation factor Tu (EF-Tu) (SEQ IDNO: 45), cochaperonin GroES-chaperonin GroEL (SEQ ID NO: 39),thioredoxin reductase (SEQ ID NO: 43), phosphoglycerate mutase (SEQ IDNO: 38), peroxiredoxin (SEQ ID NO: 40), glyceraldehyde 3-phosphatedehydrogenase (SEQ ID NO: 42), L-2,3-butanediol dehydrogenase (SEQ IDNO: 41), fructose bisphosphate aldolase (SEQ ID NO: 44), superoxidedismutase (SEQ ID NO: 37), and the like, but are not limited to these(WO2006/028063, European Patent No. 1697525).

The substitution of a stronger promoter can be combined with increasingthe copy number of the sucE1 and mdh genes.

Expression of sucE1 and mdh can also be enhanced by modifying a factorinvolved in expression control such as an operator or a repressor, orligating a strong terminator (Hamilton et al., Journal of Bacteriology171: 4617-4622). Furthermore, as disclosed in WO00/18935, a promoter canbe strengthened by making several nucleotide substitutions fornucleotides in the promoter region of a target gene so as to make thesequence closer to a consensus sequence. For example, the −35 region canbe replaced with TTGACA or TTGCCA, and the −10 region can be replacedwith TATAAT and TATAAC. In addition, it is known that the translationefficiency of mRNA is significantly affected by substitution of severalnucleotides in the spacer sequence between the ribosome-binding site(RBS) and the translation initiation codon, in particular, the sequenceimmediately upstream of the initiation codon, and therefore, such asequence can be modified.

Expression of a gene can also be enhanced by extending the survival timeof the mRNA or by preventing degradation of an enzyme protein in cells.An expression control sequence, such as a promoter, which is upstream ofthe sucE1 or mdh gene can also be identified by using a promoter searchvector or gene analysis software such as GENETYX. Expression of thesucE1 gene and the mdh gene can be enhanced by such promotersubstitution or modification. An expression control sequence can besubstituted by using, for example, a temperature-sensitive plasmid.Examples of temperature-sensitive plasmids for coryneform bacteriainclude p48K, pSFKT2 (for these, Japanese Patent Laid-open No.2000-262288), pHSC4 (French Patent Laid-open No. 1992-2667875 andJapanese Patent Laid-open No. 5-7491), and the like. These plasmids areautonomously replicable at least at 25° C., but are not autonomouslyreplicable at 37° C., in coryneform bacteria. Escherichia coli AJ12571which harbors pHSC4 was deposited at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of Trade and Industry (currently, InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, 305-5466) on Oct. 11, 1990 and assigned an accessionnumber of FERM P-11763, and this deposit was then converted to aninternational deposit under the provisions of Budapest Treaty on Aug.26, 1991 and assigned an accession number of FERM BP-3524.

Modifying an expression control sequence can be combined with increasingcopy numbers of the sucE1 gene and mdh gene.

Impartation of Other Properties

The bacterium in accordance with the presently disclosed subject mattercan be modified so that the lactate dehydrogenase (LDH) activity isdecreased, in addition enhancing expression of sucE and mdh. Theexpression “modified so that lactate dehydrogenase activity isdecreased” can mean that the LDH activity is lower than that of a strainin which the LDH is unmodified, such as a parent strain or a wild-typestrain. The lactate dehydrogenase activity per cell can be decreased to10% or lower as compared to that of an unmodified strain. The “decrease”can include the complete deletion of the activity. Reduction of the LDHactivity can be confirmed by measuring the LDH activity by a knownmethod (Kanarek, L. and Hill, R. L., 1964, J. Biol. Chem., 239:4202). Acoryneform bacterium in which the LDH activity is decreased andexpression of the sucE1 and mdh genes are enhanced can be obtained by,for example, transforming a bacterium with a disrupted LDH gene with arecombinant vector containing the sucE1 and mdh genes, as described inthe examples. However, either the modification for reducing the LDHactivity or the modification for enhancing expression of the sucE1 andmdh genes can be performed first.

In order to decrease the activity of LDH, a mutation that decreases theintracellular activity of LDH can be introduced into the LDH gene on thechromosome by a usual mutagenesis method. For example, the gene codingfor LDH on the chromosome can be deleted, or an expression controlsequence such as a promoter and/or the Shine-Dalgarno (SD) sequence canbe modified by gene recombination. Furthermore, a mutation which resultsin an amino acid substitution (missense mutation), a stop codon(nonsense mutation), or a frame shift mutation that adds or deletes oneor two nucleotides into the LDH coding region on the chromosome can beintroduced, or a part of the gene, or the entire gene, can be deleted(Journal of Biological Chemistry 272:8611-8617 (1997)). Furthermore, theLDH activity can also be decreased by mutating the LDH gene by deletingthe coding region, and replacing the normal, or native LDH gene on thechromosome with the mutant LDH gene by homologous recombination or thelike (Japanese Patent Laid-open No. 11-206385). Alternatively, atransposon or IS factor can be introduced into the genes, or the SacBgene can be used (Schafer, A. et al., Gene, 145 (1994) 69-73).

Gene substitution utilizing homologous recombination has already beenestablished, and other methods include using a linear DNA, using aplasmid containing a temperature sensitive replication origin (U.S. Pat.No. 6,303,383, Japanese Patent Laid-open No. 5-7491), and the like.

In addition, in a process for disrupting a gene as described above,levansucrase can be used as a marker for gene recombination (Schafer, A.et al., Gene, 145 (1994) 69-73).

The bacterium can also be modified so that the pyruvate carboxylase (PC)activity is enhanced, in addition to the enhanced expression of thesucE1 and mdh genes (Japanese Patent Laid-open No. 11-196888). Theexpression “modified so that pyruvate carboxylase activity is enhanced”means that the PC activity is higher than that of an unmodified strainsuch as a wild-type strain or a parent strain. The PC activity can bemeasured by, for example, by measuring the decrease of NADH. A bacteriumwith enhanced expression of the sucE1, mdh and PC genes can be producedby overexpressing the sucE1, mdh and PC genes in the same manner as thatdescribed in Japanese Patent Laid-open No. 11-196888.

As the PC gene, a gene for which the nucleotide sequence is alreadydetermined, or a gene obtained by isolating a DNA fragment encoding aprotein having the PC activity from a chromosome of a microorganism,animal, plant, or the like and determining the nucleotide sequence, canbe used. After the nucleotide sequence is determined, a gene synthesizedon the basis of that sequence can also be used.

As the PC gene, for example, a PC gene derived from, or native to, acoryneform bacterium such as Corynebacterium glutamicum(Peters-Wendisch, P. G. et al., 1998, Microbiology, vol. 144:915-927)(SEQ ID NO: 9) can be used. Furthermore, so long as the functions of theencoded PC, for example, being involved in carbon dioxide fixation, arenot substantially degraded, the PC gene can be a mutant or modifiedgene.

The PC genes from bacteria other than Corynebacterium glutamicum, aswell as from other microorganisms, animals, and plants can also be used.In particular, the reported sequences of PC genes derived frommicroorganisms, animals, and plants are described below (citations areindicated in brackets), and they can be obtained by hybridization oramplification by PCR of the ORF regions:

Human [Biochem. Biophys. Res. Comm., 202, 1009-1014, (1994)]

Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779, (1993)]

Rat [GENE, 165, 331-332, (1995)]

Yeast: Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315,(1991)], Schizosaccharomyces pombe [DDBJ Accession No.; D78170]

Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]

Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]

The PC gene can be enhanced in the same manner as for enhancingexpressions of the sucE1 and mdh genes as described above.

The bacterium can also be modified so that the acetyl-CoA hydrolase(ACH) activity is decreased, in addition to enhancing expression ofsucE1 and mdh (WO2005/113744).

The “acetyl-CoA hydrolase (ACH) activity” can mean an activity forcatalyzing the reaction to generate acetic acid from acetyl-CoA andwater, and it is known that the amount of the by-product acetic acid canbe decreased by reducing the ACH activity. The expression “modified sothat activity of acetyl-CoA hydrolase is decreased” can mean that theactivity of acetyl-CoA hydrolase is lower than the specific activity ofan unmodified strain such as a parent strain or a wild-type strain. TheACH activity per cell can be decreased to 50% or less, 30% or less inanother example, or 10% or less in another example, as compared to thatof an unmodified strain. The activity of acetyl-CoA hydrolase can bedetermined by the method of Gergely, J., et al. (Gergely, J., Hele, P. &Ramkrishnan, C. V. (1952) J. Biol. Chem. 198 p323-334). The term“decreased” can mean the complete deletion of the activity. Thecoryneform bacterium with decreased ACH activity, and enhancedexpression of the sucE1 and mdh genes, can be obtained by disrupting theACH gene in the bacterium, and transforming the bacterium with arecombinant vector containing the sucE1 and mdh genes. However, eitherthe modification for reducing the ACH activity or the modification forenhancing expressions of the sucE1 gene and the mdh gene can beperformed first.

The bacterium can also be modified so that either or both of theactivities of phosphotransacetylase (PTA) and acetate kinase (ACK)is/are decreased in addition to the enhanced expression of the sucE1gene and the mdh gene. The phosphotransacetylase (PTA) activity can meanan activity for catalyzing the reaction to generate acetyl phosphate bytransferring phosphate to acetyl-CoA (EC:2.3.1.8), and the acetatekinase (ACK) activity can mean an activity for catalyzing the reactionto generate acetic acid from acetyl phosphate and ADP (EC:2.7.2.1). Theexpression “phosphotransacetylase (PTA) activity is decreased” can meanthat the PTA activity is lower than that of a strain in which the PTA isunmodified. Although it is sufficient that the PTA activity per cell isdecreased so that it is lower than that of a strain in which the PTO isunmodified or a wild-type strain, it can be decreased to 50% or less, or10% or less in another example, as compared to the activity of a strainin which the PTA is unmodified or a wild-type strain. The PTA activitycan also be completely deleted. The decrease of the PTA activity can beconfirmed by measuring the PTA activity by the method of Klotzsch et al.(Klotzsch H. R., Meth. Enzymol., 12, 381-386 (1969)). Furthermore, theexpression “acetate kinase (ACK) activity is decreased” can mean thatthe ACK activity is lower than that of a strain in which the ACKunmodified. Although it is sufficient that the ACK activity per cell isdecreased to be lower than that of a strain in which the ACK isunmodified or a wild-type strain, it can be decreased to 50% or less,10% or less in another example, as compared to that of a strain in whichthe ACK is unmodified or a wild-type strain. The ACK activity can becompletely deleted. The decrease in the ACK activity can be confirmed bymeasuring the ACK activity by the method of Irwin A. Rose (Rose, I. A.,Meth. Enzymol., 1, 591-595 (1955)).

The coryneform bacterium in which either the activities of PTA and ACKare decreased, and expression of the sucE1 and mdh genes are enhancedcan be obtained by, for example, producing a bacterium in which eitherthe PTA or ACK gene is disrupted, and transforming the bacterium with arecombinant vector containing the sucE1 and mdh genes. However, eitherthe modification for reducing either the PTA or ACK activity or themodification for enhancing expressions of the sucE1 gene and the mdhgene can be performed first.

The bacterium can be modified so that the pyruvate oxidase (POX)activity is decreased, in addition to increasing the expression of sucE1and mdh (WO2005/113745).

The “pyruvate oxidase activity” can mean an activity for catalyzing thereaction to generate acetic acid from pyruvic acid and water. Theexpression “modified so that activity of pyruvate oxidase hydrolase isdecreased” means that the POX activity is lower than that of a strain inwhich POX is unmodified. The POX activity per cell can be decreased to50% or less, 30% or less in another example, 10% or less in anotherexample, as compared to that of an unmodified strain. The term“decreased” can include the complete deletion of the activity. Examplesof coryneform bacteria which can act as a control for comparison of theactivity include, for example, Brevibacterium lactofermentum ATCC 13869as a wild-type strain, and the Brevibacterium lactofermentum Δldh strainas an unmodified strain. The POX activity can be confirmed by measuringthe activity by the method of Chang et al. (Chang Y. and Cronan J. E.JR, J. Bacteriol., 151, 1279-1289 (1982)). The coryneform bacterium inwhich the POX activity is decreased, and the expression of the sucE1 andmdh genes are enhanced, can be obtained by disrupting the POX gene inthe bacterium, and transforming the bacterium with a recombinant vectorcontaining the sucE1 and mdh genes. However, either the modification fordecreasing the POX activity or the modification for enhancing expressionof the sucE1 gene and the mdh gene can be performed first.

Furthermore, when the organic acid is succinic acid, the bacterium canbe modified so that the activity of succinate dehydrogenase (SDH) isenhanced (Japanese Patent Laid-open No. 2005-095169), in addition to theenhanced expression of sucE1 and mdh. The expression “activity ofsuccinate dehydrogenase is enhanced” means that the activity of SDH ishigher than that of an unmodified strain such as a wild-type strain or aparent strain. The activity of SDH can be measured by measuring adecrease in NADH. A coryneform bacterium with enhanced expression of thesucE1, mdh, and SDH genes can be produced by overexpressing the sucE1,mdh, and SDH genes in the same manner as that described in JapanesePatent Laid-open No. 11-196888.

As the SDH gene, a previously reported SDH gene, or a gene obtained byisolating a DNA fragment coding for a protein having the SDH activityfrom a chromosome of a microorganism, animal or plant, and determiningthe nucleotide sequence, can be used. Moreover, after the nucleotidesequence is determined, a gene synthesized on the basis of thedetermined sequence can also be used. The sequences of the sdh operon ofCorynebacterium glutamicum (GenBank accession Nos. NCg10359 (sdhC),NCg10360 (sdhA), NCg10361 (sdhB)), and the sdh operon of Brevibacteriumflavum (Japanese Patent Laid-open No. 2005-095169, European PatentLaid-open No. 1672077) have been disclosed.

Furthermore, when the organic acid is malic acid or fumaric acid, thebacteria can be modified so that the activity of succinate dehydrogenase(SDH) is decreased, in addition to enhanced expression of sucE1 and mdh.It is known that, in Escherichia coli, if the activity of succinatereductase is insufficient, fumaric acid accumulation increases underanaerobic conditions (Journal of Industrial Microbiology & Biotechnology(2002) 28, 325-332). The expression “activity of succinate dehydrogenaseis decreased” means that the activity of SDH is lower than that of anunmodified strain such as a wild-type strain or a parent strain. Thesuccinate dehydrogenase activity per cell can be decreased to 10% orless of that of an unmodified strain. The activity of SDH can bemeasured by the aforementioned method.

The bacterium can have one of the “other properties” described above, ortwo or more of them.

Method for Producing an Organic Acid

The above-described bacterium which has an ability to produce an organicacid and has been modified to have enhanced expression of the sucE1 geneand mdh gene, or a product obtained by processing the bacterium, isallowed to act on an organic raw material in a reaction mixturecontaining carbonate ions, bicarbonate ions, or carbon dioxide gas toproduce the organic acid, and the organic acid is collected.

In the one example of the method, by culturing the microorganism in areaction mixture containing carbonate ions, bicarbonate ions, or carbondioxide gas, and an organic raw material, proliferation of themicroorganism and production of the organic acid are simultaneouslyattained. In this example, a medium can be the reaction mixture.Proliferation of the microorganism and production of the organic acidcan be simultaneously attained, or there can be a period during theculture when proliferation of the microorganism mainly occurs, and aperiod during the culture when production of the organic acid mainlyoccurs.

In another example, by allowing cells to proliferate in a medium in thepresence of carbonate ions, bicarbonate ions, or carbon dioxide gas, andan organic raw material, and thereby allowing the cells to act on theorganic raw material in the medium, an organic acid is produced. In thisexample, a product obtained by processing the cells of the bacterium canalso be used. Examples of a product obtained by processing the cellsinclude, for example, immobilized cells, which can be obtained with theuse of acrylamide, carragheenan, or the like, disrupted cells,centrifugation supernatant of the disrupted product, a fraction obtainedby partial purification of the supernatant by ammonium sulfatetreatment, or the like.

Although the bacteria can be obtained on a solid medium such as agarmedium by slant culture, bacteria previously cultured in a liquid medium(seed culture) are also an example.

When an aerobic bacterium or a facultative anaerobic bacterium is used,the bacterial cells can be cultured under aerobic conditions first, andthen used for the organic acid production reaction. Furthermore,although microaerobic induction is required for imparting an organicacid-producing activity to the cells when the cells are cultured underconventional aerobic conditions, it is not necessary. However,microaerobic induction can be performed. When microaerobic induction isperformed, it can be performed for a period of 15 hours or shorter, 10hours or shorter in another example, or 4 hours or shorter in anotherexample, although the period can differ depending on the condition ofthe bacterium and degrees of microaerobic condition.

As the medium used for the culture, typical microorganism culturemediums can be used. For example, a typical medium obtained by addingnatural nutrients such as meat extract, yeast extract, and peptone, to acomposition that includes inorganic salts such as ammonium sulfate,potassium phosphate, and magnesium sulfate can be used.

In the aforementioned first example, the carbon source, which is addedto the medium, also serves as the organic raw material for theproduction of the organic acid.

In the aforementioned second example, after the culture, the cells arecollected by centrifugation, membrane separation, or the like, and usedin the organic acid production reaction.

The organic raw material is not particularly limited so long as a carbonsource, which the chosen microorganism can assimilate to produce anorganic acid, is used. However, fermentable carbohydrates includingcarbohydrates such as galactose, lactose, glucose, fructose, glycerol,sucrose, saccharose, starch and cellulose, polyalcohols such asglycerin, mannitol, xylitol and ribitol, and the like are usually used.Among these, glucose, fructose and glycerol are examples.

Furthermore, a saccharified starch solution, molasses, or the likecontaining the fermentable carbohydrates can also be used. Thefermentable carbohydrates can be used independently or in combination.Although the concentration of the aforementioned organic raw material isnot particularly limited, it is more advantageous when the concentrationis as high as possible within such a range that the culture of themicroorganism and production of the organic acid are not inhibited. Inthe aforementioned first example, concentration of the organic rawmaterial in the medium is generally in the range of 5 to 30% (w/v), or10 to 20% (w/v) in another example. Furthermore, in the aforementionedsecond example, the concentration of the organic raw material in thereaction mixture is generally in the range of 5 to 30% (w/v), or 10 to20% (w/v) in another example. Furthermore, additional organic rawmaterial can be added as its concentration decreases with the progressof the reaction.

The aforementioned reaction mixture containing carbonate ions,bicarbonate ions, or carbon dioxide gas and the organic raw material isnot particularly limited, and it can be, for example, a medium forculturing bacteria, or it can be a buffer such as phosphate buffer. Thereaction mixture can be an aqueous solution containing a nitrogensource, inorganic salts, and the like. The nitrogen source is notparticularly limited so long as it is a nitrogen source which the chosenmicroorganism can assimilate to produce an organic acid, and specificexamples include various organic or inorganic nitrogen compounds such asammonium salts, nitrates, urea, soybean hydrolysate, casein degradationproducts, peptone, yeast extract, meat extract, and corn steep liquor.As the inorganic salts, various phosphates, sulfates, and metallic saltssuch as those of magnesium, potassium, manganese, iron, and zinc can beused. If necessary, growth-promoting factors including vitamins such asbiotin, pantothenic acid, inositol, and nicotinic acid, nucleotides,amino acids and the like can be added. In order to suppress foaming atthe time of the reaction, an appropriate amount of commerciallyavailable antifoam can be added to the medium.

pH of the reaction mixture can be adjusted by adding sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesiumhydroxide, or the like. Since the pH for the reaction is usually 5 to10, or 6 to 9.5 in another example, the pH of the reaction mixture isadjusted to be within the aforementioned range with an alkalinesubstance, carbonate, urea, or the like even during the reaction, ifneeded.

As the reaction mixture, water, buffer, medium or the like is used, buta medium is particular example. The medium can contain, for example, theaforementioned organic raw material, and carbonate ions, bicarbonateions, or carbon dioxide gas, and the reaction can be performed under ananaerobic condition. The carbonate or bicarbonate ions can be suppliedfrom magnesium carbonate, sodium carbonate, sodium bicarbonate,potassium carbonate, or potassium bicarbonate, which can also be used asa neutralizing agent. However, if necessary, carbonate or bicarbonateions can also be supplied from carbonic acid or bicarbonic acid or saltsthereof or carbon dioxide gas. Specific examples of the salts ofcarbonic acid or bicarbonic acid include, for example, magnesiumcarbonate, ammonium carbonate, sodium carbonate, potassium carbonate,ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate, and thelike. Carbonate ions or bicarbonate ions can be added at a concentrationof 0.001 to 5 M, 0.1 to 3 M in another example, or 1 to 2 M in anotherexample. When carbon dioxide gas is present, it can be present in theamount of 50 mg to 25 g, 100 mg to 15 g in another example, or 150 mg to10 g in another example, per liter of the solution.

The optimal growth temperature of the bacterium is generally in therange of 25 to 35° C. The reaction temperature is generally in the rangeof 25 to 40° C., or in the range of 30 to 37° C. in another example. Theamount of bacterial cells in the reaction mixture can be, although it isnot particularly limited, 1 to 700 g/L, 10 to 500 g/L in an example, or20 to 400 g/L in another example. The reaction time can be 1 to 168hours, or 3 to 72 hours in another example. The reaction can beperformed batch-wise or on a column

The bacterial culture can be performed under aerobic conditions.Alternatively, the organic acid production reaction can be performedunder aerobic conditions, microaerobic conditions, or anaerobicconditions. For the reaction under microaerobic conditions or anaerobicconditions, the reaction can be performed in a sealed reaction vesselwithout aeration, by supplying an inert gas such as nitrogen gas to thereaction mixture, by supplying an inert gas containing carbon dioxidegas to the reaction mixture, and the like.

The organic acid that accumulates in the reaction mixture (culturemedium) can be separated and purified from the reaction mixture in aconventional manner. Specifically, solids such as bacterial cells can beremoved by centrifugation, filtration, or the like, and then theresulting solution can be desalted with an ion exchange resin or thelike. The organic acid can be separated and purified from the solutionby crystallization or column chromatography.

Furthermore, when the target organic acid is succinic acid, after thesuccinic acid is produced, a polymerization reaction can be carried outby using the succinic acid to produce a polymer containing succinicacid. In recent years, with the increase of environmentally friendlyindustrial products, polymers prepared from raw materials of plantorigin have been attracting attention. Succinic acid can be convertedinto polymers such as polyesters and polyamides and used (JapanesePatent Laid-open No. 4-189822). Specific examples of succinicacid-containing polymers include succinic acid polyesters obtainable bypolymerizing a diol such as butanediol, ethylene glycol and succinicacid, succinic acid polyamides obtainable by polymerizing a diamine suchas hexamethylenediamine and succinic acid, and the like. In addition,succinic acid and succinic acid-containing polymers, and compositionscontaining these can be used for food additives, pharmaceutical agents,cosmetics, and the like.

EXAMPLES

Hereinafter, the present invention will be explained more specificallywith reference to the following non-limiting examples.

Example 1 Construction of a Strain in which the sucE1 and mdh Genes haveBeen Enhanced

<Construction of a Plasmid for Enhancement of sucE1>

An sucE1 gene fragment in which the native promoter is replaced with thethioredoxin reductase (TRR) promoter was obtained by crossover PCR usingsynthetic DNAs designed by referring to the nucleotide sequences aroundthe TRR and the sucE1 gene of the genome sequence of Corynebacteriumglutamicum ATCC 13032, which is publicly available (GenBank DatabaseAccession No. NC_(—)003450).

A sucE1 fragment containing the native promoter was amplified by PCRusing the chromosome of Brevibacterium flavum MJ-233 as the template andthe primers shown in SEQ ID NOS: 17 and 18. For PCR, Pyrobest DNAPolymerase (Takara Bio Inc.) was used, and the target PCR product wasobtained by incubating at 94° for 3 minutes once, and then repeating acycle of denaturation at 94° C. for 30 seconds, annealing at 60° C. for30 seconds, and extension at 72° C. for 1 minute 30 times. The PCRproduct was treated with Sse8387I, and the product was inserted intopVK9 at the Sse8387I site to construct a plasmid pVK9sucE1 carryingsucE1 gene containing the native promoter. By treating this plasmid withXbaI and BstXI, a part of the ORF of the sucE1 gene containing thenative promoter can be excised.

Furthermore, a fragment (A) containing the N-terminal sequence of thesucE1 gene was amplified by PCR using the chromosome of Brevibacteriumflavum MJ-233 as the template and the primers shown in SEQ ID NOS: 19and 20. Separately, a thioredoxin reductase (TRR) promoter fragment (B)was amplified by PCR using the chromosome of Brevibacterium flavumMJ-233 as the template and the primers shown in SEQ ID NOS: 21 and 22.In the PCR of both cases, PrimeSTAR HS DNA Polymerase (TaKaRa) was used,incubation was performed once at 98° for 2 minutes, and then a cycle ofdenaturation at 98° C. for 10 seconds, annealing at 55° C. for 5seconds, and extension at 72° C. for 20 seconds was repeated 30 times toobtain the target PCR products (A) and (B). The sequences of SEQ ID NOS:19 and 22 are complementary to each other.

Then, a fragment containing the N-terminal sequence of sucE1 in whichthe native promoter is replaced with the TRR promoter was constructed bycrossover PCR using the fragments (A) and (B) as the templates and theprimers shown in SEQ ID NOS: 20 and 21. In the PCR, PrimeSTAR HS DNAPolymerase (Takara Bio Inc.) was used, incubation was performed once at98° for 2 minutes, and then a cycle of denaturation at 98° C. for 10seconds, annealing at 55° C. for 5 seconds, and extension at 72° C. for40 seconds was repeated 30 times to obtain the target PCR product. ThePCR product was purified in a conventional manner, treated with XbaI andBstXI, and then inserted into pVK9sucE1 at the XbaI and BstXI sites toconstruct a sucE1 amplification plasmid, pVK9:PTRR-sucE1, in which thenative promoter was replaced with the TRR promoter.

pVK9 is a shuttle vector obtained by blunt-ending the Avail site ofpHSG299 (Takara Bio), and inserting a fragment, obtained by excising aregion autonomously replicable in coryneform bacteria contained in pHK4(Japanese Patent Laid-open No. 5-007491) with BamHI and KpnI andblunt-ending the excised region, into the above pHSG299 at theblunt-ended AvaII site.

<Construction of a Plasmid for Enhancement of mdh>

An mdh gene fragment in which the native promoter is replaced with theelongation factor Tu (ET-Tu) promoter was obtained by crossover PCRusing synthetic DNAs designed by referring to nucleotide sequencesaround EF-Tu and the mdh gene of the genome sequence of Corynebacteriumglutamicum ATCC 13032, which is publicly available (GenBank DatabaseAccession No. NC_(—)003450), as primers.

Specifically, an mdh fragment containing the native promoter wasamplified by PCR using the chromosome of Brevibacterium flavum MJ-233 asthe template and the primers shown in SEQ ID NOS: 23 and 24. In the PCR,Pyrobest DNA Polymerase (Takara Bio Inc.) was used, incubation wasperformed once at 94° for 2 minutes, and then a cycle of denaturation at94° C. for 30 seconds, annealing at 57° C. for 30 seconds, and extensionat 72° C. for 1 minute and 30 seconds was repeated 30 times to obtainthe target PCR product. The PCR product was treated with XbaI andSse8387I, and then inserted into pVK9 at the XbaI and Sse8387I sites toconstruct a plasmid pVK9mdh carrying mdh containing the native promoter.A part of the ORF of mdh containing the native promoter can be excisedfrom this plasmid by treating the plasmid with BamHI and MluI.

Furthermore, a fragment (C) containing an N-terminal sequence of mdh wasamplified by PCR using the chromosome of Brevibacterium flavum MJ-233 asthe template and the primers shown in SEQ ID NOS: 25 and 26. Separately,an EF-Tu promoter fragment (D) was amplified by PCR using the chromosomeof Brevibacterium flavum MJ-233 as the template and the primers shown inSEQ ID NOS: 27 and 28. In both PCR, PrimeSTAR HS DNA Polymerase (TakaraBio Inc.) was used, incubation was performed once at 98° for 2 minutes,and then a cycle of denaturation at 98° C. for 10 seconds, annealing at55° C. for 5 seconds, and extension at 72° C. for 20 seconds wasrepeated 30 times to obtain the target PCR products (C) and (D). Thesequences of SEQ ID NOS: 25 and 28 are complementary to each other.

Then, a fragment containing the N-terminal sequence of mdh in which thenative promoter is replaced with the EF-Tu promoter was constructed bycrossover PCR using the fragments (C) and (D) as the templates and theprimers shown in SEQ ID NOS: 27 and 26. In the PCR, PrimeSTAR HS DNAPolymerase (Takara Bio Inc.) was used, incubation was performed once at98° for 2 minutes, and then a cycle of denaturation at 98° C. for 10seconds, annealing at 55° C. for 5 seconds, and extension at 72° C. for40 seconds was repeated 30 times to obtain the target PCR product. ThePCR product was purified in a conventional manner, treated with BamHIand MluI, and then inserted into pVK9mdh at the BamHI and MluI sites toconstruct a mdh amplification plasmid, pVK9:PEFTu-mdh, in which thenative promoter was replaced with the EF-Tu promoter.

<Construction of a Plasmid for Enhancement of sucE1 and mdh>

A plasmid carrying both the sucE1 and mdh genes was constructed asfollows.

First, an mdh gene fragment in which the native promoter was replacedwith the ET-Tu promoter was obtained by crossover PCR using syntheticDNAs designed by referring to nucleotide sequences around EF-Tu and themdh gene of the genome sequence of Corynebacterium glutamicum ATCC13032, which is publicly available (GenBank Database Accession No.NC_(—)003450), as primers.

A fragment (G) containing the ORF of mdh was amplified by PCR using thechromosome of Brevibacterium flavum MJ-233 as the template and theprimers shown in SEQ ID NOS: 25 and 29. In the PCR, PrimeSTAR HS DNAPolymerase (Takara Bio Inc.) was used, incubation was performed once at98° for 2 minutes, and then a cycle of denaturation at 98° C. for 10seconds, annealing at 55° C. for 5 seconds, and extension at 72° C. for1 minute was repeated 30 times to obtain the target PCR product (G).Separately, an EF-Tu promoter fragment (H) was amplified by PCR usingthe chromosome of Brevibacterium flavum MJ-233 as the template and theprimers shown in SEQ ID NOS: 30 and 28. In the PCR, PrimeSTAR HS DNAPolymerase (Takara Bio Inc.) was used, incubation was performed once at98° for 2 minutes, and then a cycle of denaturation at 98° C. for 10seconds, annealing at 55° C. for 5 seconds, and extension at 72° C. for20 seconds was repeated 30 times to obtain the target PCR product (H).The sequences of SEQ ID NOS: 25 and 27 are complementary to each other.

Then, a fragment containing ORF of mdh in which the native promoter wasreplaced with the EF-Tu promoter was constructed by crossover PCR usingthe fragments (G) and (H) as the templates and the primer shown in SEQID NOS: 29 and 30. In the PCR, PrimeSTAR HS DNA Polymerase (Takara BioInc.) was used, incubation was performed once at 98° for 2 minutes, andthen a cycle of denaturation at 98° C. for 10 seconds, annealing at 55°C. for 5 seconds, and extension at 72° C. for 1 minute and 20 secondswas repeated 30 times to obtain the target PCR product. The PCR productwas purified in a conventional manner, treated with SpeI and Sse8387I,and then inserted into pVK9:PTRR-sucE1 at the SpeI and Sse8387I sites toconstruct a plasmid carrying both sucE1 and mdh,pVK9:PTRR-sucE1+PEFTu-mdh.

A plasmid carrying both sucE1 and mdh can be constructed in the samemanner by using chromosomal DNA of a strain other than the MJ-233 strainsuch as the Corynebacterium glutamicum AJ110655 strain as the template.

<Construction of ldh Gene-Disrupted Strain>

(A) Cloning of a Fragment to Disrupt the ldh Gene

A gene fragment of the ldh gene deficient in the ORF was obtained bycrossover PCR using synthetic DNAs designed by referring to thenucleotide sequence around NCg12817 of the genome sequence ofCorynebacterium glutamicum ATCC 13032, which is publicly available(GenBank Database Accession No. NC_(—)003450), as primers. PCR wasperformed using the chromosomal DNA of Brevibacterium lactofermentum2256 strain (ATCC 13869) as the template and the synthetic DNAs of SEQID NOS: 31 and 32 as primers to obtain an amplification product of anN-terminal sequence of the ldh gene. Separately, to obtain anamplification product of a C-terminal sequence of the ldh gene, PCR wasperformed by using the chromosomal DNA of Brevibacterium lactofermentum2256 strain as the template and the synthetic DNAs of SEQ ID NOS: 33 and34 as primers. In the PCR, Pyrobest DNA Polymerase (Takara Bio Inc.) wasused, and each target PCR product was obtained by performing incubationat 94° for 3 minutes once, and then repeating a cycle of denaturation at94° C. for 30 seconds, annealing at 60° C. for 30 seconds, and extensionat 72° C. for 1 minute 30 times. The sequences of SEQ ID NOS: 32 and 33are complementary to each other. Then, to obtain an ldh gene fragment inwhich the internal sequence is deleted, the above gene products of theN-terminal and C-terminal regions of ldh were mixed in approximatelyequimolar amounts, and PCR was performed using the mixture as thetemplate and synthetic DNAs of SEQ ID NOS: 35 and 36 as primers toobtain an amplification product in which of ORF of ldh is deleted. Inthe PCR, Pyrobest DNA Polymerase (Takara Bio Inc.) was used, and thetarget PCR product was obtained by performing incubation at 94° for 3minutes once, and then repeating a cycle of denaturation at 94° C. for30 seconds, annealing at 58° C. for 30 seconds, and extension at 72° C.for 2 minute 30 times. The PCR product was purified in a conventionalmanner, then digested with BamHI, and inserted into a vector pBS4S forgene disruption (WO2007/046389) at the BamHI site to obtain a plasmidfor ldh gene disruption, pBS4SΔldh56. The construction of this plasmidis shown in FIG. 1.

(B) Production of ldh-Disrupted Strain

The pBS4SΔldh56 obtained in (A) described above does not contain aregion that enables autonomous replication in a cell of coryneformbacteria. Therefore, when a coryneform bacterium is transformed withthis plasmid, the strain in which the plasmid is integrated into thechromosome by homologous recombination appears as a transformant,although it occurs at an extremely low frequency. The Brevibacteriumlactofermentum 2256 strain was transformed with a high concentration ofthe plasmid pBS4SΔldh56 by the electric pulse method, and thetransformed cells were applied onto CM-Dex agar medium (5 g/L ofglucose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g/L ofKH₂PO₄, 0.4 g/L of MgSO₄.7H₂O, 0.01 g/L of FeSO₄.7H₂O, 0.01 g/L ofMnSO₄.7H₂O, 3 g/L of urea, 1.2 g/L of soybean hydrolysate, pH 7.5 (KOH)containing 1.5% of agar) containing 50 μg/ml of kanamycin, and culturedat 31.5° C. for about 24 hours. The plasmid used for the transformationof MJ-233 was obtained by transforming a dam methylase-deficient strain,Escherichia coli SCS110 strain (Stratagene), with pBS4SAΔldh56, andextracting the plasmid from this strain. In the strains of colonies thatappeared, the kanamycin resistance gene and the sacB gene derived fromthe plasmid were inserted into the genome as a result of homologousrecombination between the ldh gene fragment on the plasmid and the ldhgene on the genome of Brevibacterium flavum MJ-233 strain.

Then, these single recombinants were cultured overnight at 31.5° C. inthe CM-Dex liquid medium not containing kanamycin, then appropriatelydiluted, applied to 10% sucrose-containing Dex-S10 medium (100 g/L ofsucrose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g/L ofKH₂PO₄, 0.4 g/L of MgSO₄.7H₂O, 0.01 g/L of FeSO₄.7H₂O, 0.01 g/L ofMnSO₄.4H₂O, 3 g/L of urea, 1.2 g/L of soybean hydrolysate, 10 μg/L ofbiotin, pH 7.5 (KOH) containing 1.5% of agar) not containing kanamycin,and cultured at 31.5° C. for about 24 hours to obtain clones which areresistant to sucrose. These strains no longer expressed the normal sacBgene, and included strains in which pBS4SAΔldh56 was eliminated by thesecond homologous recombination. Furthermore, the strains which hadundergone the second homologous recombination included a strain in whichthe ldh gene was replaced with the deletion-type gene derived frompBS4SΔldh56 and a strain in which the ldh gene had reverted back to thewild-type gene. Whether the ldh gene is the mutant or the wild-type canbe easily confirmed by extracting chromosomal DNA from cells obtained byculture on the Dex-S10 agar medium, and detecting the ldh gene in thechromosomal DNA by PCR. Among the double recombinant strains, afteramplification using the primers for PCR amplification of the ldh gene(SEQ ID NOS: 31 and 34), a strain which provided a PCR product smallerthan the PCR product obtained by using the chromosomal DNA of the MJ-233strain was determined to be an ldh-deficient strain, and was used in thefollowing experiments. The ldh-deficient strain was designated asAJ110655.

<Construction of sucE1-Amplified Strain, mdh-Amplified Strain, andsucE1+mdh-Amplified Strain>

The Corynebacterium glutamicum AJ110655 strain was transformed withpVK9:PTRR-sucE1, pVK9:PEFTu-mdh, pVK9:PTRR-sucE1+PEFTu-mdh obtainedabove, and pVK9, by the electric pulse method, applied to the CM-Dexagar medium containing 25 μg/ml of kanamycin, and cultured at 31.5° C.for about 24 hours to obtain strains into which each of the plasmidswere introduced. The colonies that appeared were purified, and plasmidswere extracted in a conventional manner to confirm introduction of eachtarget plasmid.

Example 2 Succinic Acid Production with the sucE1- and mdh-AmplifiedStrain

Using strains obtained by introducing each of pVK9, the plasmid forenhanced sucE1 gene alone (pVK9:PTRR-sucE1), the plasmid for enhancedmdh gene alone (pVK9:PEFTu-mdh), and the plasmid for enhanced sucE1 andmdh genes together (pVK9:PTRR-sucE1+PEFTu-mdh), into the Corynebacteriumglutamicum AJ110655 strain, and the culture for succinic acid productionwas performed as follows. Cells of each strain obtained by culturing thestrain on a CM-Dex plate medium were inoculated into 20 ml of a seedmedium (25 g/L of glucose, 1.4 g/L of (NH₄)₂SO₄, 0.5 g/L of KH₂PO₄, 0.5g/L of K₂HPO₄, 0.5 g/L of MgSO₄.7H₂O, 4 g/L of urea, 0.02 g/L ofFeSO₄.7H₂O, 0.02 g/L of MnSO₄.7H₂O, 200 μg/L of biotin, 200 μg/L ofVB1.HCl, 1 g/L of yeast extract, 1 g/L of casamino acid, 25 mg/L ofkanamycin), and cultured at 31.5° C. in a Sakaguchi flask under anaerobic conditions with shaking for about 5 hours (aerobic culture) or16 hours (microaerobic induction culture).

Then, 700 μl of the seed medium was isolated, and immediately mixed with700 μl of a main medium (200 g/L of glucose, 30 g/L of Na sulfite,filtrated and mixed with 143 g/L as the final concentration of magnesiumcarbonate subjected to hot air sterilization) contained in a micro tube(Eppendorf tube), and the culture was performed at 31.5° C. withshaking. The culture was terminated after 48 hours, and the amount ofsuccinic acid which was produced was analyzed by liquid chromatography.Two of Shim-pack SCR-102H (Shimadzu) connected in series were used asthe column, and a sample was eluted at 40° C. with 5 mMp-toluenesulfonic acid. The eluate was neutralized with 20 mM Bis-Trisaqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA,and succinic acid was quantified by measuring electric conductivity withCDD-10AD (Shimadzu).

When the microaerobically cultured cells obtained when microaerobicinduction was performed, i.e., obtained with the seed culture of 16hours, were used, the amount of succinic acid which accumulated per cellwas equivalent in all the transformants. Alternatively, when theaerobically cultured cells obtained when the microaerobic induction wasnot performed, i.e., obtained with the seed culture of 5 hours, wereused, only the pVK9:PTRR-sucE1+PEFTu-mdh-introduced strain produced anamount of succinic acid higher than those obtained with the cellsobtained with microaerobic induction, whereas the pVK9-introducedstrain, pVK9:PTRR-sucE1-introduced strain, and pVK9:PEFTu-mdh-introducedstrain did not produce succinic acid during the main culture. Theresults are shown in Table 1.

From these results, it was demonstrated that production of succinic acidcan be imparted in a strain in which expression of both the sucE1 andmdh genes are enhanced, when the strain is cultured under aerobicconditions, and not under microaerobic conditions, and is effective forfermentative production of succinic acid.

TABLE 1 Succinic acid accumulation amount (g/L/O.D.620 = 1) AerobicallyMicroaerobically Strain cultured cells cultured cells AJ110655/pVK9 0.61.6 AJ110655/pVK9: PTRR-sucE1 0.7 1.8 AJ110655/pVK9: PEFTu-mdh 0.5 1.9AJ110655/pVK9: PTRR-sucE1 + 2.0 1.7 PEFTu-mdh

Explanation of Sequence Listing

SEQ ID NO: 1: Primer for sucE1 gene amplification

SEQ ID NO: 2: Primer for sucE1 gene amplification

SEQ ID NO: 3: Nucleotide sequence of sucE1 gene of B. flavum MJ-233

SEQ ID NO: 4: Amino acid sequence encoded by the sucE1 gene mentionedabove

SEQ ID NO: 5: sucE1 gene of C. glutamicum ATCC 13032

SEQ ID NO: 6: Amino acid sequence encoded by the sucE1 gene mentionedabove

SEQ ID NO: 7: Nucleotide sequence of sucE1 gene derived from C.efficiens YS314

SEQ ID NO: 8: Amino acid sequence encoded by the sucE1 gene mentionedabove

SEQ ID NO: 9: Nucleotide sequence of PC gene of C. glutamicum

SEQ ID NO: 10: Amino acid sequence encoded by the PC gene mentionedabove

SEQ ID NO: 11: Nucleotide sequence of sucE1 gene of C. diphtheriaegravis NCTC 13129

SEQ ID NO: 12: Amino acid sequence encoded by the sucE1 gene mentionedabove

SEQ ID NO: 13: Nucleotide sequence of mdh gene of B. flavum MJ-233

SEQ ID NO: 14: Amino acid sequence encoded by the mdh gene mentionedabove

SEQ ID NO: 15: Primer for mdh gene amplification

SEQ ID NO: 16: Primer for mdh gene amplification

SEQ ID NO: 17: Primer for amplification of sucE1 fragment containingnative promoter

SEQ ID NO: 18: Primer for amplification of sucE1 fragment containingnative promoter

SEQ ID NO: 19: Primer for amplification of fragment (A) containingN-terminal sequence of sucE1

SEQ ID NO: 20: Primer for amplification of fragment (A) containing the Nterminal sequence of sucE1

SEQ ID NO: 21: Primer for amplification of TRR promoter fragment (B)

SEQ ID NO: 22: Primer for amplification of TRR promoter fragment (B)

SEQ ID NO: 23: Primer for amplification of mdh fragment containingnative promoter

SEQ ID NO: 24: Primer for amplification of mdh fragment containingnative promoter

SEQ ID NO: 25: Primer for amplification of fragment (C) or (G)containing N-terminal sequence of mdh

SEQ ID NO: 26: Primer for amplification of fragment (C) containingN-terminal sequence of mdh

SEQ ID NO: 27: Primer for amplification of EF-Tu promoter fragment (D)

SEQ ID NO: 28: Primer for amplification of EF-Tu promoter fragment (D)or (H)

SEQ ID NO: 29: Primer for amplification of fragment (G) containing mdhgene

SEQ ID NO: 30: Primer for amplification of EF-Tu promoter fragment (H)

SEQ ID NO: 31: Primer for production of fragment for ldh gene disruption(N-terminal sequence)

SEQ ID NO: 32: Primer for Production of Fragment for Ldh Gene Disruption(N-terminal sequence)

SEQ ID NO: 33: Primer for production of fragment for ldh gene disruption(C-terminal sequence)

SEQ ID NO: 34: Primer for production of fragment for ldh gene disruption(C-terminal sequence)

SEQ ID NO: 35: Primer for production of fragment for ldh gene disruption

SEQ ID NO: 36: Primer for production of fragment for ldh gene disruption

SEQ ID NO: 37: Nucleotide sequence of superoxide dismutase gene promoterof C. glutamicum ATCC 13032

SEQ ID NO: 38: Nucleotide sequence of phosphoglycerate mutase genepromoter of C. glutamicum ATCC 13032

SEQ ID NO: 39: Nucleotide sequence of GroES-GroEL operon promoter of C.glutamicum ATCC 13032

SEQ ID NO: 40: Nucleotide sequence of peroxiredoxin gene promoter of C.glutamicum ATCC 13032

SEQ ID NO: 41: Nucleotide sequence of butanediol dehydrogenase genepromoter of C. glutamicum ATCC 13032

SEQ ID NO: 42: Nucleotide sequence of glyceraldehyde 3-phosphatedehydrogenase gene promoter of C. glutamicum ATCC 13032

SEQ ID NO: 43: Nucleotide sequence of thioredoxin reductase genepromoter of C. glutamicum ATCC 13032

SEQ ID NO: 44: Nucleotide sequence of fructose bisphosphate aldolasegene promoter of C. glutamicum ATCC 13032

SEQ ID NO: 45: Nucleotide sequence of EF-Tu gene promoter of B. flavumMJ-233

INDUSTRIAL APPLICABILITY

According to the method of the present invention, an organic acid can bequickly and highly efficiently produced from an organic raw material byusing aerobically cultured cells without need of microaerobic induction.The obtained organic acid can be used for food additives,pharmaceuticals, cosmetics, and the like. Moreover, organicacid-containing polymers can also be produced by performing apolymerization reaction using the obtained organic acid as a rawmaterial.

While the invention has been described in detail with reference toexemplary embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

1. A method for producing an organic acid comprising: A) allowing asubstance to act on an organic raw material in a reaction mixturecontaining carbonate ions, bicarbonate ions, or carbon dioxide gas,wherein the substance is selected from the group consisting of: i) abacterium which has an ability to produce an organic acid and has beenmodified to have enhanced expression of the sucE1 and mdh genes, ii) aproduct obtained by processing the bacterium of i), and iii)combinations thereof, and B) collecting the organic acid.
 2. The methodaccording claim 1, wherein expression of the sucE1 and mdh genes underaerobic conditions is enhanced by 1.5 times or more as compared to anunmodified strain.
 3. The method according to claim 1, wherein thebacterium is selected from the group consisting of coryneform bacteria,Bacillus bacteria, and Rhizobium bacteria.
 4. The method according toclaim 1, wherein enhanced expression is obtained by a method selectedfrom the group consisting of i) increasing the copy number of the sucE1gene and/or the mdh gene, ii) modifying an expression control sequenceof the sucE1 gene and/or the mdh gene, iii) replacing a promoter of thesucE1 gene and/or the mdh gene with a stronger promoter, and iv)combinations thereof.
 5. The method according to claim 4, wherein thestronger promoter is a constitutive expression promoter.
 6. The methodaccording to claim 4, wherein the stronger promoter is a promoter of agene that encodes a protein selected from the group consisting ofelongation factor Tu, cochaperonin GroES-chaperonin GroEL, thioredoxinreductase, phosphoglycerate mutase, peroxiredoxin, glycerol-3-phosphatedehydrogenase, 2,3-butanediol dehydrogenase, fructose bisphosphatealdolase, and superoxide dismutase.
 7. The method according to claim 1,wherein the sucE1 gene is selected from the group consisting of: (a) aDNA comprising the nucleotide sequence of the numbers 571 to 2187 of SEQID NO: 3, and (b) a DNA which hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence of the numbers 571 to 2187 ofSEQ ID NO: 3 under stringent conditions, and the DNA improves theability of the bacterium to produce succinic acid when expression of theDNA is enhanced in the bacterium.
 8. The method according to claim 1,wherein the mdh gene is selected from the group consisting of: (a) a DNAcomprising the nucleotide sequence of the numbers 301 to 1287 of SEQ IDNO: 13, and (b) a DNA which hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence of the numbers 301 to 1287 ofSEQ ID NO: 13 under stringent conditions, and codes for a protein havingmalate dehydrogenase activity.
 9. The method according to claim 1,wherein the bacterium has been further modified so that lactatedehydrogenase activity is decreased to 10% or less as compared tolactate dehydrogenase activity in an unmodified strain.
 10. The methodaccording to claim 1, wherein the bacterium has been further modified sothat pyruvate carboxylase activity is enhanced.
 11. The method accordingto claim 1, wherein the organic acid is succinic acid.
 12. A method forproducing a succinic acid-containing polymer comprising: A) producingsuccinic acid by the method according to claim 1, and B) polymerizingthe succinic acid.
 13. The method according to claim 11, wherein thebacterium has been further modified so that succinate dehydrogenaseactivity is enhanced.
 14. The method according to claim 1, wherein theorganic acid is malic acid or fumaric acid.
 15. The method according toclaim 14, wherein the bacterium has been further modified so thatsuccinate dehydrogenase activity is decreased to 10% or less as comparedto succinate dehydrogenase activity in an unmodified strain.